NDC80 promotes proliferation and metastasis
of colon cancer cells
X.K. Xing1, H.Y. Wu2, H.L. Chen1 and H.G. Feng1
Department of Life Science and Technology, Xinxiang Medical University,
Xinxiang, China
2
Department of Forensic Biology, Xinxiang, China
1
Corresponding author: X.K. Xing
E-mail: biyingxiao@163.com
Genet. Mol. Res. 15 (2): gmr.15028312
Received December 21, 2015
Accepted February 26, 2016
Published May 6, 2016
DOI http://dx.doi.org/10.4238/gmr.15028312
ABSTRACT. Chromosome instability is a common feature of tumor
cells, and may be an important mechanism in tumor formation. Nuclear
division cycle 80 (NDC80) is closely associated with the stability of
chromosomes. Therefore, we investigated the relationship between
NDC80 and development of colon cancer using a range of methods.
Western blotting and immunohistochemistry were employed to determine
the expression of this protein in different colon cells and tissues, cell
proliferation was measured with an MTT assay, levels of proliferating
cell nuclear antigen were examined by immunofluorescence, and cell
migration was observed using wound healing tests. Our results showed
that the expression of NDC80 in colon cancer cells (CACO2, HCT8,
HCT116, and SW480) and tissues (from 20 patients) was higher than
that in controls. Moreover, cell proliferation and migration rates were
elevated in cells transfected with NDC80 compared to control groups.
In summary, NDC80 promotes the proliferation and metastasis of colon
cancer cells, and may constitute a new target for gene therapy in treating
Genetics and Molecular Research 15 (2): gmr.15028312
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X.K. Xing et al.
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this disease. Combined with clinicopathological grading, measurement
of positive NDC80 expression may be helpful in diagnosing and
estimating the prognosis of colon cancer patients.
Key words: Chromosome instability; Colon cancer; Hec1; Metastasis;
Nuclear division cycle 80; Proliferation
INTRODUCTION
Colon cancer is a common malignancy of the digestive tract, usually occurring in the
rectum and its junction with the sigmoid colon. It is one of the most common malignant tumors
in western Europe, North America, and other developed countries (Zou et al., 2014). Owing to
its high recurrence rate, it represents a serious public health problem (Bishnupuri et al., 2010). At
present, treatment is principally based on surgery and radiotherapy. This approach is effective for
patients in the early stages of this disease, but achieves poor outcomes for those with advanced
cancer and microcarcinoma. With the development of molecular biology and related sciences,
gene targeting therapy has become a research topic of significant interest in tumor treatment
(Zhang et al., 2014; Maus et al., 2015). Gene targeting relies on the selection of a gene for
treatment, therefore, the identification of a specific target affecting tumors is important.
Chromosome instability is a common feature of tumor cells, and may be an important
mechanism in tumor formation (Venkitaraman, 2001; Luo et al., 2011; Venkitaraman, 2014).
Accurate regulation of the binding and dynamic changes of microtubules is essential for
chromosome stability. The kinetochore is a complex of multiple proteins that plays a key role
in this process. Nuclear division cycle 80 (NDC80), known in humans as highly expressed in
cancer (Hec1), is a nuclear protein rich in coiled-coil motifs. It was discovered by Durfee et
al. (1993) using yeast two-hybrid technology and the C-terminus of the retinoblastoma protein
(RB) to screen a B lymphocyte complementary DNA library. NDC80 complexes are important
to the spindle assembly checkpoint, and participate in the regulation of mitosis (Ciferri et al.,
2008; Alushin et al., 2012). In this study, we analyzed the biological function of NDC80 in
the proliferation and metastasis of colon cancer cells, and provided new reference data and
experimental support for colon cancer-targeting gene therapy.
MATERIAL AND METHODS
Materials
Normal human intestinal epithelial NCM460 cells and the colon cancer cell lines SW480,
HCT8, HCT116, and CACO2 were purchased from the Chinese Academy of Medical Sciences
tumor hospital. Between January 2015 and November 2015, 20 colon cancer patients (42-58 years
old, twelve men and eight women) were recruited from the First Affiliated Hospital of Xinxiang
Medical College, and underwent surgical resection of tumorous and adjacent tissue. Pathological
examination showed that of these, seven cases were highly differentiated adenocarcinoma, six
were moderately differentiated adenocarcinoma, and seven were undifferentiated carcinoma.
Twelve cases demonstrated lymph node metastasis, while eight did not. Six patients were at
stage I, five at stage II, five at stage III, and four at stage IV (according to the tumor-nodemetastasis staging of colon cancer). All patients gave their written informed consent.
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NDC80 promotes colon cancer cell growth and metastasis
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Main reagents
Fetal bovine serum (FBS) was purchased from Sijiqing Hangzhou Biological Engineering
Materials Company (Hangzhou, China), Dulbecco’s modified Eagle’s medium (DMEM) and
trypsin were sourced from Gibco (Waltham, MA, USA), Lipofectamine was provided by Invitrogen
(Carlsbad, CA, USA), mouse anti-human NDC80 and rabbit anti-human b-actin antibodies were
purchased from Chemicon (Billerica, MA, USA), mouse anti-human proliferating cell nuclear
antigen (PCNA) antibody was manufactured by Beijing Jinqiao Biotechnology (Beijing, China),
and enhanced chemiluminescence reagent was produced by Pierce (Waltham, MA, USA). An
ECLIPSE 80i fluorescence microscope was also used (Nikon, Tokyo, Japan).
Detection of NDC80 protein expression in different cells by western blot
Normal human intestinal epithelial NCM460 cells, and colon cancer SW480, HCT8,
HCT116, and CACO2 cells were cultured in DMEM containing 10% FBS at 37°C in a 5% CO2
atmosphere. Total protein was extracted and separated by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis, before being transferred to a cellulose nitrate membrane. Skim milk powder
was used to block the membrane for 1 h at 37°C. Anti-NDC80 antibody was then added and
incubated overnight with shaking at 4°C. The membrane was then exposed to horseradish
peroxidase-labeled secondary antibody for 1 h at 37°C. Bound antibodies were visualized by
chemiluminescence, and images were scanned with a Bio-Rad ChemiDoc MP (Hercules, CA,
USA) gel imaging system to obtain gray values using the Quantity One software.
Detection of NDC80 protein expression in different colon tissues by
immunohistochemistry
Healthy and cancerous paraffin-embedded colon tissue was cut into 5-mm slices and
immunohistochemically stained. The expression of NDC80 was observed under a microscope.
Gene transfection
Experiments comprised control, empty plasmid, and NDC80-transfected groups.
When SW480 cells reached 60 to 70% confluence, the serum volume fraction of the medium
was reduced to 5%. Lipofectamine (100 mL) was then mixed with 1 mg plasmid for 20 min,
and added to the culture medium. After 6 h, this was replaced with medium containing 10%
serum, and 48 h later, culture medium containing 200 ng/L G418 was used to select transfected
cells. After two weeks, 24-well plates were used to clone and amplify selected cells.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for
measurement of cell proliferation
Cell density was adjusted to 2 x 104/mL and 100 mL of cells per well was added to
a 96-well plate, with six wells for each experimental group. MTT reagent (20 mL/well) was
added to cell cultures of various ages (1-6 days), and incubated for 4 h at 37°C in a CO2
incubator. The culture medium was then carefully removed and 150 mL dimethyl sulfoxide
added to wells, before shaking the plate for 10 min. Absorbance at 570 nm was measured in
each well using a microplate reader, and a cell growth curve was generated.
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X.K. Xing et al.
PCNA expression detected by immunofluorescence assay
Cell were fixed for 30 min with 4% formaldehyde at room temperature, then incubated
with phosphate-buffered saline (PBS) containing 1% Triton X-100, before being blocked with
goat serum for 30 min. After removal of the serum, PCNA antibody was incubated with cells at
4°C overnight. Cells were then exposed to fluorescein isothiocyanate (FITC)-labeled secondary
antibody at 37°C for 45 min. After being washed with PBS and mounted, cells were observed
under a fluorescence microscope. PCNA expression was determined as follows: PCNA-positive
rate = FITC-positive cells/total cells x 100%. Twenty fields of view were examined each sample.
Wound healing
When cell confluence approached 80%, a 10-mL plastic pipette tip was used to scratch
the culture gently, and then pictures were taken every 12 h under a microscope. Cultures were
observed for 72 h, and the width of the scratch was measured.
Statistical analysis
Data were analyzed by the t-test using SPSS 13.0 (SPSS Inc., Chicago, IL, USA), and
P values < 0.05 were considered statistically significant.
RESULTS
NDC80 protein expression in different colon cells
NDC80 protein was highly expressed in CACO2, HCT8, HCT116, and SW480 colon
cancer cells, but levels in normal human intestinal epithelial NCM460 cells were significantly
decreased in comparison (Figure 1).
Figure 1. Expression of NDC80 protein in various colon cells measured by western blot.
NDC80 protein expression in different colon tissues
NDC80-positive tissues were identified by yellow or brownish yellow staining. Of the
20 samples of normal colonic tissue, seven (35%) were positive, while among the 20 colon
cancer specimens, 15 (75%) were positive. This difference was statistically significant (P <
0.05; Figure 2).
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NDC80 promotes colon cancer cell growth and metastasis
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Figure 2. Expression of NDC80 protein in different colon tissues visualized by immunohistochemical staining
(200X magnification). A. Normal colon tissue; B. colon carcinoma.
MTT-based cell growth curve
Proliferation of SW480 cells in the NDC80-transfected group was greater than that of
the control groups at the same time points, and this difference was statistically significant (P
< 0.05). The growth rate of SW480 cells did not significantly differ between the control and
empty plasmid groups (P > 0.05; Figure 3).
Figure 3. Growth curves of cells in each group.
Cellular PCNA expression
PCNA-positive rates in the control, empty plasmid, and NDC80-transfected groups
were 24.2 ± 3.4, 30.4 ± 5.6, and 45.1 ± 5.5%, respectively. Compared to the control and empty
plasmid groups, PCNA expression in the NDC80-transfected group was significantly higher
(P < 0.05, Figure 4).
SW480 cell migration
72 h later,the widths of the wounds created in cultures of SW480 cells in the control,
empty plasmid, and NDC80-transfected groups were 89.27 ± 6.23, 80.34 ± 7.16, and 64.77 ±
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X.K. Xing et al.
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9.18 mm, respectively. SW480 cells in the NDC80-transfected group gradually migrated to the
middle of the wound, shortening the distance between the two sides. In the control and empty
vector groups, there were significantly fewer SW480 cells in the wound area, and the distance
between the two sides was greater than that observed with the NDC80-transfected group, to a
statistically significant degree (P < 0.05, Figure 5).
Figure 4. PCNA expression in each group was visualized by immunofluorescent staining (100X magnification). A.
control group, B. empty plasmid group, C. NDC80-transfected group.
Figure 5. Wound healing in each group (100X magnification). A. control group, B. empty plasmid group, C.
NDC80-transfected group.
CONCLUSIONS
NDC80 is a nuclear protein with a coiled-coil structure, consisting of 642 amino acids.
Its N-terminus contains at least three functional binding sites: a predicted nuclear localization
sequence, a Nek2 phosphorylation site, and a specific RB interaction site. The C-terminus
incorporates a series of seven leucine heptad repeat sequences, which play an important role
in protein interactions (DeLuca et al., 2005). NDC80 is highly conserved in eukaryotic cells.
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NDC80 is a cell cycle protein expressed during cell division. In particular, levels of
this protein significantly increase from S to M phase, and it has a regulatory function during the
G2/M phase (Sundin et al., 2011). NDC80 expression in all cells undergoing division is high,
and it is not expressed in terminally differentiated cells. This protein maintains chromosome
stability (Wei et al., 2011; Wei et al., 2014), and in cells with abnormal NDC80 expression,
spindle checkpoint dysfunction, abnormal chromosome separation, and cell cycle disorder
may occur, perhaps ultimately leading to tumor formation (Alushin et al., 2012; Tang and
Toda, 2015). NDC80 assembly plays an important role in the spindle checkpoint process.
Nuclear aneuploidy results from unstable chromosomes, and is a common feature of
tumor cells. A previous study found that aspects of tumor behavior, such as recurrence and
invasion, correlate with the number of aneuploid cells. The development of aneuploidy is an
important mechanism in tumor development. (Shinmura et al., 2014). Colon cancer is a common
malignant tumor of the digestive system associated with development, lifestyle, genetic factors,
and colorectal adenoma. Cytogenetic analysis has revealed most colon cancers to be aneuploid.
Many variations of chromosome number exist, including sub-diploidy, pseudodiploidy, and
tetraploidy (Baker and van Deursen, 2010). The distribution of aneuploidy in a tumor is an
important indicator of progression and invasion; treatment effectiveness and prognosis for
aneuploid tumors are significantly worse than those seen in relation to diploid tumors.
This study found that NDC80 protein was highly expressed in SW480, HCT8, HCT116,
and CACO2 colon cancer cells, and the NDC80-positive rate was significantly higher in colon
cancer specimens than in normal colon tissues. Moreover, MTT and PCNA assays revealed
faster cell proliferation in the NDC80-transfected group, while wound healing tests showed
greater migration ability in cells transfected with NDC80 compared to controls. Our results
demonstrate that NDC80 is significantly associated with invasion and metastasis in colon
cancer. This protein may promote the occurrence of aneuploidy, leading to the development
of this disease. NDC80 may therefore represent a new target for colon cancer gene therapy.
The results of this study provide a new theoretical basis for gene therapy in colorectal cancer.
However, the number of samples in our study was limited, and the role of NDC80 in colon
cancer development was examined using only in vitro experiments, limiting the strength of
any conclusions drawn. In future work, in vivo tests involving expanded sample sizes will be
carried out to verify the present results.
Conflicts of interest
The authors declare no conflict of interest.
ACKNOWLEDGMENTS
Research supported by research grants from the Key Research Project of Henan Province
(#15A180020) and the National Natural Science Foundation of China (#U1304819, 81401519).
REFERENCES
Alushin GM, Musinipally V, Matson D, Tooley J, et al. (2012). Multimodal microtubule binding by the Ndc80 kinetochore
complex. Nat. Struct. Mol. Biol. 19: 1161-1167. http://dx.doi.org/10.1038/nsmb.2411
Baker DJ and van Deursen JM (2010). Chromosome missegregation causes colon cancer by APC loss of heterozygosity.
Cell Cycle 9: 1711-1716. http://dx.doi.org/10.4161/cc.9.9.11314
Genetics and Molecular Research 15 (2): gmr.15028312
©FUNPEC-RP www.funpecrp.com.br
X.K. Xing et al.
8
Bishnupuri KS, Luo Q, Sainathan SK, Kikuchi K, et al. (2010). Reg IV regulates normal intestinal and colorectal cancer
cell susceptibility to radiation-induced apoptosis. Gastroenterology 138: 616-626, 626.e1-626.e2. http://dx.doi.
org/10.1053/j.gastro.2009.10.050
Ciferri C, Pasqualato S, Screpanti E, Varetti G, et al. (2008). Implications for kinetochore-microtubule attachment from
the structure of an engineered Ndc80 complex. Cell 133: 427-439. http://dx.doi.org/10.1016/j.cell.2008.03.020
DeLuca JG, Dong Y, Hergert P, Strauss J, et al. (2005). Hec1 and nuf2 are core components of the kinetochore outer plate
essential for organizing microtubule attachment sites. Mol. Biol. Cell 16: 519-531. http://dx.doi.org/10.1091/mbc.
E04-09-0852
Durfee T, Becherer K, Chen PL, Yeh SH, et al. (1993). The retinoblastoma protein associates with the protein phosphatase
type 1 catalytic subunit. Genes Dev. 7: 555-569. http://dx.doi.org/10.1101/gad.7.4.555
Luo F, Poulogiannis G, Ye H, Hamoudi R, et al. (2011). Mutant K-ras promotes carcinogen-induced murine colorectal
tumourigenesis, but does not alter tumour chromosome stability. J. Pathol. 223: 390-399. http://dx.doi.org/10.1002/
path.2790
Maus MK, Hanna DL, Stephens CL, Astrow SH, et al. (2015). Distinct gene expression profiles of proximal and distal
colorectal cancer: implications for cytotoxic and targeted therapy. Pharmacogenomics J. 15: 354-362. http://dx.doi.
org/10.1038/tpj.2014.73
Shinmura K, Kurabe N, Goto M, Yamada H, et al. (2014). PLK4 overexpression and its effect on centrosome regulation
and chromosome stability in human gastric cancer. Mol. Biol. Rep. 41: 6635-6644. http://dx.doi.org/10.1007/s11033014-3546-2
Sundin LJ, Guimaraes GJ and Deluca JG (2011). The NDC80 complex proteins Nuf2 and Hec1 make distinct contributions
to kinetochore-microtubule attachment in mitosis. Mol. Biol. Cell 22: 759-768. http://dx.doi.org/10.1091/mbc.E1008-0671
Tang NH and Toda T (2015). MAPping the Ndc80 loop in cancer: A possible link between Ndc80/Hec1 overproduction
and cancer formation. BioEssays 37: 248-256. http://dx.doi.org/10.1002/bies.201400175
Venkitaraman AR (2001). Chromosome stability, DNA recombination and the BRCA2 tumour suppressor. Curr. Opin.
Cell Biol. 13: 338-343. http://dx.doi.org/10.1016/S0955-0674(00)00217-9
Venkitaraman AR (2014). Tumour suppressor mechanisms in the control of chromosome stability: insights from BRCA2.
Mol. Cells 37: 95-99. http://dx.doi.org/10.14348/molcells.2014.2346
Wei R, Ngo B, Wu G and Lee WH (2011). Phosphorylation of the Ndc80 complex protein, HEC1, by Nek2 kinase
modulates chromosome alignment and signaling of the spindle assembly checkpoint. Mol. Biol. Cell 22: 3584-3594.
http://dx.doi.org/10.1091/mbc.E11-01-0012
Wei X, Gao C, Luo J, Zhang W, et al. (2014). Hec1 inhibition alters spindle morphology and chromosome alignment in
porcine oocytes. Mol. Biol. Rep. 41: 5089-5095. http://dx.doi.org/10.1007/s11033-014-3374-4
Zhang D, Huang Y and Wang H (2014). [Advances of driver gene and targeted therapy of non-small cell lung cancer].
Zhongguo Fei Ai Za Zhi 17: 750-754.
Zou AM, Wang HF, Zhu WF, Wang FX, et al. (2014). Effect of RNAi-mediated silencing of Livin gene on biological
properties of colon cancer cell line LoVo. Genet. Mol. Res. 13: 3832-3841. http://dx.doi.org/10.4238/2014.May.16.8
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